The fine art of preparing membrane transport proteins for biomolecular simulations: Concepts and practical considerations

Abstract

Molecular dynamics (MD) simulations have developed into an invaluable tool in bimolecular research, due to the capability of the method in capturing molecular events and structural transitions that describe the function as well as the physiochemical properties of biomolecular systems. Due to the progressive development of more efficient algorithms, expansion of the available computational resources, as well as the emergence of more advanced methodologies, the scope of computational studies has increased vastly over time. We now have access to a multitude of online databases, software packages, larger molecular systems and novel ligands due to the phenomenon of emerging novel psychoactive substances (NPS). With so many advances in the field, it is understandable that novices will no doubt find it challenging setting up a protein-ligand system even before they run their first MD simulation. These initial steps, such as homology modelling, ligand docking, parameterization, protein preparation and membrane setup have become a fundamental part of the drug discovery pipeline, and many areas of biomolecular sciences benefit from the applications provided by these technologies. However, there still remains no standard on their usage. Therefore, our aim within this review is to provide a clear overview of a variety of concepts and methodologies to consider, providing a workflow for a case study of a membrane transport protein, the full-length human dopamine transporter (hDAT) in complex with different stimulants, where MD simulations have recently been applied successfully.

Keywords: Computational modelling; Membrane protein simulations; Molecular docking; Molecular dynamics; Protein structure preparation; Small molecule parameterization.

Figure 1.. Two-dimensional schematic representation of the topology of the human dopamine transporter (hDAT).

Colored regions indicate the transmembrane (TM) domains that are embedded in the lipid bilayer. Areas outside of this region appear as not coloured and are either extracellular or intracellular loops or the N- and C- termini that both reside on the intracellular side.

Figure 2.. A schematic of the multi-step process for molecular docking

(1) simple ‘template’ recognition; (2) sequence alignment; (3) model building for the intended ‘target’, which is based on the 3D structure of the ‘template’; (4) model refinement, analysis of alignments, gap deletions as well as additions and finally (5) model validation.

Figure 3.. hDAT homology model predicted from the dDAT crystal structure

(a) The dDAT crystal structure (Protein Database (PDB) ID: 4M48) exhibiting the LeuT-like structure fold. (b) A model of the hDAT tertiary structure based on the alignment in [110] with predicted extracellular loop 2 (EL2) and N- and C- termini regions indicated. (c) Superimposition of (a) and (b).

Figure 4.. Workflow for preparation for IFD docking of three compounds.

(a) The 3D coordinates of cocaine were retrieved from the PBD (PDB ID: 4XP4) and then prepared in Schrödinger to add hydrogens and a positive charge to the nitrogen on the tropane group, (b) 5-IT was built in Schrödinger by modifying the 3D coordinates of amphetamine (PDB ID: 4XP9); hydrogens were added and a positive charge was added to the amine nitrogen (c) the 3D coordinates for D2PM were retrieved from PubChem: Compound CID: 704537; hydrogens were added and a positive charge was added to the nitrogen in the pyrrole ring. In the last panel for each compound you can see that each of these distinct ligands (shown in red) occupies a binding pocket that is deeply buried in the transporter structure and overlaps with the binding site of the substrate dopamine. Selected central binding site residues from each ligand are shown in yellow and labelled respectively. The internal sodium and chloride ions are shown in magenta and purple, respectively.

Figure 5.. A schematic workflow of the docking procedure in Schrödinger.

Individual descriptions for the ligand and protein preparation as well as details for the IFD protocol are given. The 3D coordinates for the protein are either obtained from the PDB or homology modeling, while the 3D coordinates for the ligand are either obtained from PubChem or built manually (Figure 4).